Group A streptococci bind to mucin and human pharyngeal cells through sialic acid-containing receptors

Abstract

The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with (125)I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified alpha2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.

FIG. 1

Binding of group A streptococci to bovine submaxillary mucin. The figure shows adherence of S. pyogenes D471 to mucin- and BSA-coated wells of a microtiter plate at 37 and 30°C. Wells were coated with 1 μg of mucin, blocked with BSA, and inoculated with 5 × 10⁶ bacteria. The plates were incubated for 1 h and washed, and the bacteria were desorbed with 0.5% Triton X-100 and plated. The mean ± standard deviation (error bars) was derived from triplicate wells in at least three independent experiments. P values are indicated above corresponding bars. The inset shows the adherence of strain D471 to ¹²⁵I-mucin. Increasing concentrations of labeled mucin were added to microtiter plate wells containing immobilized heat-killed bacteria (5 × 10⁶ bacteria/well) and incubated for 4 h. The wells were washed, and the counts in each well (representing bound mucin) were determined in a gamma counter. Nonspecific binding was assessed as binding to BSA-coated wells and subtracted from the total binding. Means ± standard deviations (error bars) obtained from triplicate wells in at least two independent experiments are shown.

FIG. 2

Identification of group A streptococcal cell wall proteins that bind bovine submaxillary mucin. Cell wall-associated proteins were extracted with lysin enzyme from streptococcal strains D471 and JRS75 (the isogenic M-negative mutant). Extracted proteins (40 μg of total protein/strain) and the recombinant M protein from E. coli (2 μg) were separated on an SDS–8% polyacrylamide gel and transferred to PVDF membranes. (A) Autoradiogram of blot of D471 cell wall proteins probed with ¹²⁵I-bovine mucin. (B) At left is shown an autoradiogram of blots of cell wall proteins from D471 and JRS75, as well as purified M protein, probed with ¹²⁵I-bovine mucin. The right panel shows a Western blot of extracted proteins probed with the M-protein-specific MAb 10B6.

FIG. 3

Identification of the mucin binding region of the M protein. The recombinant M protein from E. coli (5 μg) was digested with pepsin at pH 5.9 for 30 min, separated by SDS-PAGE, stained with Coomassie blue (left panel), or electroblotted onto a PVDF membrane. The right panel shows an autoradiogram of a Western blot that was probed with ¹²⁵I-bovine mucin. Following hybridization, the membrane was washed and autoradiographed for 12 h at −80°C. Untreated M protein (2 μg) from E. coli served as control. Arrows indicate the positions of the intact M protein (57 kDa) and the three peptide bands that represent the N-terminal portion (30 kDa) and the two C-terminal regions (15 and 12 kDa) of the pepsin-digested M protein.

FIG. 4

Effect of monosaccharides on the binding of group A streptococci to bovine mucin. (A) Streptococcal strain D471 (5 × 10⁷ CFU) was preincubated with 25 mM aliquots of GlcNAc, GalNAc, fucose, galactose, or NANA (sialic acid) for 30 min at 37°C. Bacteria were used to inoculate mucin-coated wells (1 μg of mucin/well inoculated with 5 × 10⁶ CFU/well). (B) Mucin-coated wells were inoculated with strain D471, which was treated with 25 mM NANA (as described above) and incubated at 37 or 30°C to assess the effect of temperature on mucin binding. In all experiments, untreated bacteria served as control. The number of adherent bacteria was determined as described in Materials and Methods. In both panels, means ± standard deviations (error bars) were derived from triplicate wells in at least two independent experiments. P values are indicated above the corresponding bars in panel B.

FIG. 5

Effect of monosaccharides on adherence and internalization of streptococci to Detroit 562 pharyngeal cells. Streptococcal strain D471 (5 × 10⁷ CFU) was preincubated with 25 mM aliquots (prepared in 10 mM Tris-HCl, pH 7.4) of fucose, GalNAc, GlcNAc, or NANA for 30 min at 37°C. Bacteria were washed and then used to infect confluent wells of Detroit 562 pharyngeal cells (5 × 10⁷ CFU/well). Untreated bacteria served as a control. The adherence and internalization assays were performed for 2.5 h at 37°C, as described in Materials and Methods. Means ± standard deviations (error bars) were derived from triplicate wells in at least two independent experiments. See Results for P values.

FIG. 6

Effect of different concentrations of sialic acid on adherence to pharyngeal cells. Streptococcal strain D471 (5 × 10⁷ CFU) was preincubated with different concentrations of sialic acid (25 to 0.5 mM) for 30 min at 37°C and then used to infect confluent wells of Detroit 562 pharyngeal cells (5 × 10⁷ CFU/well). Untreated bacteria served as control. The adherence and internalization assays were performed for 2.5 h at 37°C, as described in Materials and Methods. Means ± standard deviations (error bars) were derived from triplicate wells in at least two independent experiments. P values are indicated above the corresponding bars.

FIG. 7

Effect of sialic acid containing glycoconjugates and neuraminidase on adherence of streptococci to Detroit 562 pharyngeal cells. (A) S. pyogenes D471 and the isogenic M-negative strain JRS75 (5 × 10⁷ CFU/ml) were preincubated with sialic acid (25 mM) and used in a pharyngeal cell adherence assay as described in Materials and Methods. (B) To determine the sialylated linkages that play a role in adherence of D471 to pharyngeal cells, the bacteria (5 × 10⁷ CFU) were individually incubated for 30 min at 37°C with the following compounds: 3′SL (1.7 mM), fetuin (5 mg, 1.8 mM total sialic acid), transferrin (5 mg), and sialic acid (NANA) (25 mM); cells were then washed and used in the adherence assays. Sialylated linkages of each compound are reported in Results. Streptococci incubated with Tris-HCl (10 mM, pH 7.4) served as a control (strep. alone). (C) Strain D471 (5 × 10⁷ CFU) was preincubated with sialic acid (NANA) (2 mM) and 6′SL (1.7 mM) as described above prior to performing the adherence assay. (D) Detroit cell monolayers were treated at confluence with C. perfringens neuraminidase (as described in Materials and Methods) to determine the effect on streptococcal adherence. After treatment, the monolayers were inoculated with strain D471 (5 × 10⁷ CFU/well) and the adherence assay was performed as described. In all panels, means ± standard deviations (error bars) were derived from duplicate wells in at least two independent experiments. P values are indicated above the corresponding bars.

FIG. 8

Identification of sialylated pharyngeal cell membrane proteins which bind the streptococcal M protein. Membrane-associated proteins from pharyngeal cell line Detroit 562 were extracted and solubilized as described in Materials and Methods. Extracted proteins (50 μg of total protein/lane) were separated (in duplicate) on SDS–8% polyacrylamide gels and transferred to PVDF membranes. The left panel shows a blot probed with 25 μg of the purified M protein, and the right panel shows a blot probed with 2 μg of the sialic acid-specific lectin, TML. Bound M protein and TML were detected as described in Materials and Methods. Arrows indicate the three pharyngeal cell membrane proteins that bound to both the M protein and TML.